We propose to clone and sequence several hundred genes involved in multicellular morphogenesis to expand the reference set of characterized genes. Insertional mutagenesis will be used to tag each gene and simultaneously indicate the developmental role of the gene product. Recent advances in molecular genetic techniques in Dictyostelium make this a favored organism for detailed studies on genes involved in cellular differentiation and intercellular interactions. Development of Dictyostelium involves processes common to many multicellular organisms such as activation of genes necessary for production of intercellular signals as well as cellular response mechanisms, changes in membrane proteins involved in cell-cell adhesion, specialization of cell-types characterized by expression of specific genes, proportioning and sorting out of the cell-types to generate tissues, and terminal cellular differentiation. These processes take place within 24 hours after growth has ceased and depend on the function of about 300 genes that are dispensable for vegetative growth. Mutations in any one of these developmental genes result in visible aberrations of multicellular morphogenesis that can be recognized in clones that develop within plaques cleared in a bacterial lawn. We have found that Restriction Enzyme Mediated Integration (REMI) of linearized plasmid DNA stimulates the rate of transformation 20 to 50 fold and allows us to generate the number of transformants necessary to tag most of the developmental genes. About 0.3% of the transformants selected for integration of a plasmid vector carrying either the pyr5-6 gene or the thyA gene are unable to develop normally due to disruption of a gene at the site of integration. By screening several hundred thousand transformants, we can tag and subsequently clone most of the 300 genes necessary for development in this system. We have already collected over 50 mutant strains in which developmental genes are tagged by insertional mutagenesis. These fall into a variety of classes based on the stage at which development is blocked such as aggregation, mound formation, tip formation, slug migration, and early culmination of fruiting bodies. Other genetic screens and selections will undoubtedly produce new classes of mutants. We plan to collaborate with colleagues throughout the world in the analysis of as many important genes as possible.